The objective of this proposal is to isolate mouse mutants that can be used to study the etiology and to improve the treatment of phenylketonuria, PKU, and other related heritable defects. Such mutants are important for basic neurological and enzymological studies as well as for the applications directly related to clinical medicine. The mutations will be induced by ethylnitrosourea mutagenesis of spermatogonial stem cells and detected through a three-generation breeding scheme designed to produce homozygotes in which these recessive mutations are expressed and cause elevated levels of phenylalanine in the blood. The proposed work develops procedures that are quite general and that can be readily adapted to obtain the analogous mutants needed in studies of other human diseases or in the genetic dissection of a variety of normal mammalian physiological and developmental pathways. The ability, to generate specific new mouse mutants, and to utilize genetic strategies that previously were restricted to microorganisms or drosophila, creates completely new possibilities for mammalian genetics. To permit the greatly expanded use of mutants and to minimize the cost of animal maintenance as this new potential becomes more widely appreciated, a simple and reliable method for preserving sperm in the frozen state for subsequent use in artificial insemination will be perfected.